Adherent resorbable matrix

ABSTRACT

The present invention is directed to an adherent resorbable matrix for use in surgical applications. The adherent resorbable matrix includes a biocompatible adherent material including chitosan.

RELATED CASE INFORMATION

This application claims priority to provisional application Ser. No.61/215,875 filed May 11, 2009 and provisional application Ser. No.61/273,741 filed Aug. 7, 2009 each of which are hereby incorporated byreference herein in their entireties.

FIELD OF THE INVENTION

The present invention relates to an adherent resorbable matrix for usein surgical applications, for example neurosurgical and orthopedicapplications as a dural substitute for the repair and restoration ofdural defects in cranial and spinal surgical procedures.

BACKGROUND

Dural substitutes made of resorbable materials are known in the art.Dural substitutes for promoting meningeal tissue growth that include,for example collagen, are described in U.S. Pat. No. 5,997,895. Acommercially available resorbable matrix is DURAGEN Dural Graft Matrix(Integra LifeSciences Corporation, Plainsboro, N.J.) Resorbable matricesplaced on tissue may migrate out of place during surgery as a result offluid being present at the site of the matrix, such as blood,cerebralspinal fluid, or irrigation fluid or by contact from the surgeonor surgical instruments. It is desirable to minimize displacement of theresorbable matrix in an in vivo environment, such as during surgery.

It is therefore an object of the present invention to provide anadherent resorbable matrix that adheres to a tissue site in vivo, suchas a surgical site, which may come in contact with aqueous fluid. It isalso an object of the present invention to provide a resorbable matrixwith a material that promotes the adherence of the resorbable matrix toa tissue site in vivo which may come in contact with an aqueous fluid,such as a surgical site. These and other objects, features, andadvantages of the invention or certain embodiments of the invention willbe apparent to those skilled in the art from the following disclosureand description of exemplary embodiments.

SUMMARY

Embodiments of the present invention are directed to a resorbable matrixthat adheres for a period of time to an in vivo site which may includean aqueous fluid, such as a moist body tissue surface. The resorbablematrix includes a material that promotes the adherence of the resorbablematrix to the in vivo site and in the presence of an aqueous fluid, suchas blood, cerebralspinal fluid, or irrigation fluid. In the context ofembodiments of the present invention described herein, the terms“adherent,” “adherence” and “adhesive” are used interchangeably andindicate the property of embodiments of the present invention to resistmovement when placed on a tissue surface under influence of fluid beingpresent, force of fluid or force of contact by a surgeon.

According to certain embodiments of the present invention, duralsubstitutes are provided having increased adhesive capacity. The duralsubstitute includes a resorbable matrix with a sticky material appliedto at least one surface of the matrix. The term “sticky” indicates theproperty of the embodiments of the invention to resist movement whenplaced on a tissue surface under influence of fluid being present orforce of fluid against the dural substitute or force of contact by asurgeon, and does not necessarily indicate a tackiness of the materialas part of a dural substitute. The resorbable matrix can be a matrixsheet made of a biocompatible material, for example, a bioresorbablenatural polymer, a bioresorbable synthetic polymer or a combinationthereof. The adherent material may be a layer made from anynon-cytotoxic, biocompatible chemical or mixture of chemicals, forexample, a chemical that is water soluble and is a solid at roomtemperature in its pure state.

In accordance with another aspect of the invention, there is provided aself adhesive resorbable matrix including an adhesive material thatdemonstrates an increase in resistance to migration compared to a matrixlacking the adhesive material. The self adhesive resorbable matrixprovides a matrix for tissue regeneration that resists migration, due inpart to a polysaccharide adhesive layer. In accordance with one aspect,the self adhesive resorbable matrix aids or otherwise promotes fibrinclot formation. Additionally, the self adhesive resorbable matrixprovides an adherent regeneration matrix that remains in place andresists movement even in the absence of a fibrin clot or secondarysecuring devices such as sutures or staples.

In accordance with a further aspect of the invention, there is providedan adhesive resorbable matrix including a biocompatible matrix sheet anda biocompatible adhesive applied on at least one surface of thebiocompatible matrix sheet. In more detailed aspects, the biocompatiblematrix sheet can be a porous collagen matrix, and the biocompatibleadhesive comprises a polysaccharide material and derivatives andmodifications thereof. Polysaccharides include, but are not limited to,polysaccharides including free base moieties and salt moieties. Onesuitable example of a polysaccharide useful in the present invention ischitosan, chitosan derivatives and salts of thereof. Certain chitosanand chitosan derivatives and salts thereof, have a degree ofdeacetylation in the range of 40-99%. Certain adhesives are crosslinked,partially crosslinked or uncrosslinked as appropriate.

Embodiments of the present invention are also directed to a method ofmaking an adherent resorbable matrix including applying an adherentmaterial, such as in the form of a fluid, slurry or the like, to atleast one surface of a resorbable matrix that is to contact tissue whichmay have a moist or wet surface or otherwise be in an in vivo aqueousenvironment. The resorbable matrix with the adherent material is thentreated to remove the fluid, such as by lyophilization, to create theresorbable matrix with the adherent material thereon.

Embodiments of the present invention are also directed to a method ofusing an adherent resorbable matrix including applying the adherentresorbable matrix to tissue with a moist or wet surface or otherwise inan in vivo aqueous environment such that the resorbable matrix remainsin place, for example with little migration, for a desired period oftime.

Other features and advantages of the present invention will become moreapparent from the following detailed description of the invention.

DETAILED DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS

Embodiments of the present invention are based on a resorbable matrixcharacterized in that the resorbable matrix adheres for a period of timeto tissue, and may be referred to herein as an adherent resorbablematrix, adhesive resorbable matrix, self adherent resorbable matrix,sticky resorbable matrix or self adhesive resorbable matrix. Embodimentsof the adherent resorbable matrix include a resorbable matrix and amaterial that promotes adherence of the resorbable matrix to the tissueby providing sufficient tack to the resorbable matrix so that theresorbable matrix resists movement when placed on tissue, even undermoist, wet or aqueous conditions.

Tissues to which the adherent resorbable matrix can be applied includetissues forming the solid or semi-solid structures that make up any ofthe organs or compounds of a living organism, preferably human, such asmembranes, skin, muscles, bones, cartilage, nerves and nerve sheathes,meninges, connective tissue, blood vessels, the sclera or iris of theeye, the solid materials constituting internal organs such as liver,stomach, pancreas, intestine, kidney, thymus, uterus, testes, bladder,lung, heart and any other internal structures that are solid orsemi-solid in texture and including tissues of the gastrointestinalsystem, the parenchymal organs, the cardiovascular system, the thoracicsystem, the pulmonary system, the ear, the nose, the throat, the dentalarea, the gynecological system, the urological system, the vascularsystem, the bone system, the neurological system, the lymphatic system,the derma, the biliary system and the like.

One component of the self adhesive resorbable matrix is a porous matrixsheet made of a biocompatible material, for example, a bioresorbablenatural, semisynthetic or synthetic polymer or a combination thereof. Anadditional component of the self adhesive resorbable matrix is anadhesive or adherent material which can be in the form of a layercontacting the resorbable matrix and/or partially or wholly impregnatedinto the matrix or not, on one or both sides of the resorbable matrixwhen, for example, the resorbable matrix is in sheet form. The selfadhesive resorbable matrix can include additional biocompatiblecomponents, bioactive agents or other active ingredients.

Both the matrix and the adhesive material forming the self adhesiveresorbable matrix are preferably biocompatible and biodegradable. Uponmoistening with fluids, the matrix readily conforms to the surface ofunderlying tissues and when applied with the adhesive contacting tissue,aids in fibrin clot formation and hemostasis along with regeneration ofthe tissue. Similarly, the adhesive layer also functions to aid infibrin clot formation and/or hemostasis while facilitating the adherenceof the matrix to the tissue to prevent displacement/migration of thedevice after initial positioning and during surgical irrigation,fixation or other activities. Without wishing to be bound by theory,adhesives having cationic functional groups like protonated aminefunctional groups, such as with chitosan in acidic or neutral media,react through electrostatic interactions with anionic functional groupsin blood proteins and tissue to form a clot. Accordingly, aspects of thepresent invention include methods of inducing, aiding, promoting, and/oreffecting clot formation, such as fibrin clot formation, by applying theadherent resorbable matrix of the present invention to tissue andallowing the interaction between cationic species of the adherentresorbable matrix and anionic species in blood and tissue in a manner toform a clot.

The matrix may include any biocompatible polymer. The biocompatiblepolymer may be any naturally occurring polymer, a semisynthetic polymer,a synthetic polymer or a combination thereof. For example, the matrixcan comprise a material selected from processed animal proteins, tissuesor polysaccharides or plant derived polysaccharide or proteins, polymersderived from other living organisms, synthetic materials, orcombinations of the aforementioned materials. Useful naturally-occurringbiocompatible polymers include fibrous or globular proteins, complexcarbohydrates, glycosaminoglycans, and the like or combinations thereof.Certain naturally-occurring biocompatible polymers within the scope ofthe present invention include collagens, such as collagens of all types,cellulose, gelatin, elastin, alginic acid, hyaluronic acid, versican,desmin, microcellular proteins such as osteonectin, osteopontin,thrombospondin 1 and 2, fibrin, fibronectin, vitronectin, albumin,chitin, chitosan and the like or combinations thereof. According tocertain aspects of the present invention, naturally-occurringbiocompatible polymers are used as a component for the carrier sheetalone or in combination with other naturally-occurring biocompatiblepolymers or synthetic polymers. Exemplary biocompatible polymers includeheparin, hyaluronic acid, polyhydroxy acid, lactic acid, glycolic acid,copolymers thereof such as PGA/PLA (poly glycolic acid/poly lacticacid), hydroxybutanoic acid, cellulose, gelatin, collagen, chitin,chitosan and the like or a combination thereof. Suitable syntheticbiocompatible polymers may include, for example, 2-hydroxyethylmethacrylate, silicone rubber, poly (e-caprolactone) dimethylacrylate,polysulfone, (poly) methyl methacrylate, soluble Teflon-AF, polyethyleneterephthalate, nylon, polyvinyl alcohol, polyurethane, polyaminoacids,polydepsipeptides and the like or combinations thereof.

A particular exemplary biocompatible polymer is collagen. Collagenwithin the scope of the present invention includes collagens from anyknown class, for example, Class I, II, III, IV, etc. Sources of collageninclude mammalian sources, transgenic sources, recombinant sources, andthe like or combinations thereof. Exemplary sources include humansources and bovine sources. If the collagen source is a non-humanmammal, such as bovine corium or bovine tendon collagen, it isadvantageous to inactivate potentially pathogenic agents. According tothis aspect, the collagen is greater than 90% pure, substantially freeof all prion and viral contamination, has less than 0.03 eu/gmendotoxins, has not more than 5% fat content, has at least 10%hydroxyproline content and has not more than 5% ash content. In anexemplary embodiment of the present invention, the matrix comprises typeI bovine collagen, lyophilized into a sponge-like sheet. The collagenmatrix aids in the regeneration of the tissue and the adhesive surfacereduces the displacement of the matrix after initial positioning andduring irrigation or other activities.

The matrix may also include a mixture of biocompatible polymers thatbiodegrade at different rates. For example, the matrix may includeslow-biodegrading collagen and slow-biodegrading polymer, slowbiodegrading collagen and fast-biodegrading polymer, fast-biodegradingcollagen and slow-biodegrading polymer or fast-biodegrading collagen anda fast-biodegrading polymer. One of skill in the art will readilyunderstand based on the present disclosure that combinations of variousbiocompatible materials having various degradation rates may be combinedto achieve a desired degradation profile for a particular application.

According to certain aspects of the present invention, polymers used tomake the matrix, such as collagen, may be crosslinked, partiallycrosslinked or non-crosslinked. Crosslinking methods include those knownto persons of ordinary skill in the art including chemical crosslinking, ultraviolent radiation, dehydrothermal cross linking, and thelike or combinations of these treatments and for a time periodsufficient to achieve a desired degree of crosslinking. Cross linkingagents include carbodimide, gluteraldehyde, formaldehyde, diisocyanates,mono, di and polysaccharides, oxidized polysaccharides, enzymes such asgenipin and transglutaminase, and the like and mixtures thereof. Thedegree of crosslinking may range from about 0% (non-crosslinked) toabout 100% (fully crosslinked) or any percentage in between (partiallycrosslinked.) One of skill in the art based upon the disclosure hereinwill recognize that the degree of crosslinking affects the bioresorptionor biodegradation of the collagen. In general, the more highly crosslinked the collagen, the longer the collagen may remain within anindividual before degradation, for example, weeks, months, and years.Collagen with a low degree of crosslinking can have utility for days orweeks before degradation. Crosslinking can be used to impart a desiredstability to the collagen during the intended use of the material. Thedegree of crosslinking includes from about 0% to about 100%, about 10%to about 90%, or about 15% to about 85% of the reactive functionalgroups and any ranges and values in between these ranges whetheroverlapping or not.

The matrix can be fashioned into any desired form or shape, such as asheet for placement over tissue, a tube within which damaged tissue canbe placed or as a mold of a tissue defect site into which the matrix isto be placed. When in sheet form, the sheet should be flexible,comfortable, and of substantially uniform thickness. The sheet should beflexible and readily assume the general shape and contours of thetissues and parts of, for example, the human body to which the sheet isapplied. The sheet may also have elastic characteristics allowing it tostretch in one or two directions. The sheet should also be conformableand capable of adapting to the overall shape of the tissues and partsof, for example, the human body, via intimate contact without creatingvoids or kinks. Further, the sheet may have substantially uniformthickness across its longitudinal (y) and transverse (x) directions.

One skilled in the art would easily recognize that the thickness of thesheet would depend upon the particular application. Typically, the sheethas a thickness of about 0.001 mm to about 100 mm, from about 0.1 mm toabout 50 mm, from about 0.3 mm to about 10 mm, from about 0.5 mm toabout 10 mm, from about 1.0 mm to about 5 mm, from about 2.5 mm to about5 mm or about 3 mm and any ranges or value in between whetheroverlapping or not. The carrier sheet may have any density. One skilledin the art would recognize that the density of the sheet would dependupon the particular application. Typically, the sheet has a density ofabout 1 mg/cm³ to about 100 mg/cm³.

In one embodiment, a matrix is provided in the form of a collagensponge. The matrix can also be provided in the form of a non-wovenmatrix, felt or film according to methods known in the art. The physicalstructures of a non-woven matrix, felt or film are known to those ofskill in the art and can include varying densities, porosities, porestructure, fiber structure and the like depending upon the particularmethod of manufacture. In addition, composites of the various forms ofthe matrix can be made such as a film/sponge or a film/sponge/film. Acomposite of as many layers of a matrix material can be made as desiredand according to the desired application. In one aspect of the presentinvention, a laminate of a collagen sponge and a collagen film isprovided.

This laminate, which can be formed, e.g., by laminating a collagensponge to a collagen film with a biocompatible adhesive or polymer(including collagen), by forming a sponge on a film, or by forming afilm on a sponge, possess the elevated water impermeability andsuturability of a film, and the elevated porosity of a sponge, whichfacilitates dural tissue growth there through. Similarly, asandwich-type laminate can be provided by providing a collagen spongebetween opposing sheets of collagen film.

The matrix of the present invention includes pores of sufficient sizeand quantity to permit growing meningeal tissue to infiltrate therein.The pore size ranges, whether internal pores or surface pores, fromabout 1 μm to about 1000 μm, about 10 μm to about 500 μm, about 30 μm toabout 15.0 μm, about 50 μm to about 300 μm, or from about 50 μm to about150 μm and any range or value in between the ranges whether overlappingor not.

The porous matrix has a purity defined for medical applications as lessthan about 100 EU/g. For example, it can have endotoxin levels less thanabout 40 EU/g. The porous matrix can have a resorption time of betweenabout 0.5 days to about 2 yrs in the human body, for example, in therange of about 30 days to about 180 days.

The matrix can include biocompatible and/or bioresorbable materialsother than collagen. For example, in certain embodiments it isadvantageous to laminate the collagen matrix to a non-collagen film,such as a 50:50 dl lactide:co-glycolide polymer having a molecularweight of about 75,000 and more preferably about 100,000. Additionalsuitable polymers include, e.g., biocompatible and/or bioresorbablelactides, glycolides, and copolymers thereof, polycaprolactones,polyethylene carbonate, tyrosine polycarboronates, tyrosine polyacids,polyanhydrides and the like. The molecular weight of the polymer ispreferably about 5000 to about 500,000.

The adhesive can comprise a natural, semisynthetic or synthetic polymer,a derivative of such polymer, other biocompatible adhesive material, ora combination of two or more of the aforementioned materials. Accordingto one aspect, the biocompatible adhesive can be polycationic polymers,polyanionic polymers, nonionic polymers, salt, protein, fatty molecule,other biocompatible adhesive materials and combinations thereof. Anexample of a biocompatible adhesive material is a water soluble or apartially water soluble carbohydrate including low, medium and highmolecular weight saccharides, such as a monosaccharide, disaccharide orpolysaccharide. Such carbohydrates and saccharides include sugars,starches, gums, glycosaminoglycans, celluloses and hemicelluloses andderivatives and modifications thereof.

Adhesive materials within the scope of the present invention have amolecular weight between about 5 kDa and about 2000 kDa, about 10 kDaand about 1500 kDa, about 10 kDa and 1000 kDa, or about 50 kDa and 1500kDa. Low molecular weight (LMW) adhesive materials may have a molecularweight between about 5 kDa to about 250 kDa or from about 70 kDa toabout 150 kDa. Medium molecular weight (MMW) adhesive materials may havea molecular weight between about 250 kDa to about 750 kDa or from about300 kDa to about 700 kDa. High molecular weight (HMW) adhesive materialsmay have a molecular weight between about 750 kDa to about 2000 kDa orfrom about 700 kDa to about 1250 kDa. One of skill will recognize thatthe present adhesive material can have a molecular weight within anyranges or value within the above ranges whether overlapping or not. Oneof skill will recognize based on the present disclosure that themolecular weight of the adhesive layer material can depend on theparticular adhesive formulation and the desired adhesive property.Suitable adhesives can be applied in an amount between about 1% andabout 99%, between about 25% and about 75%, or between about 40% andabout 60% of the total weight of the adherent resorbable matrix.

Suitable adhesives according to certain embodiments include watersoluble or partly water soluble free base forms and/or salt forms ofcarbohydrates and saccharides. Examples of acceptable salts include, butare not limited to, mineral or organic acid salts of basic residues suchas amines; alkali or organic salts of basic residues such as carboxylicacids; and the like. The acceptable salts include the conventionalnon-toxic salts or the quaternary ammonium salts from non-toxicinorganic acids. For example, such conventional non-toxic salts includethose derived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric, nitric, and the like; and the saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmoic,maleic, hydroxymaleic, phenylacetic, glutamine, benzoic, salicylic,sulfanilic, 2-acteoxybenzoic, fumaric, toluenesulfonic, methanesulfonic,ethane disulfonic, oxalic, isethionic, and the like. Specifically, theacceptable salts can include those salts that naturally occur in vivo ina mammal. According to certain embodiments, exemplary salts are thoseformed from acids having a pKa of between about 1.5 and 8.5. Exemplarysalts also include those formed from acids having a pKa of between about2.5 and about 6.5 or between about 4.0 and 6.0. A particular exemplarysalt includes that formed from acetic acid which has a pKa of about 4.7.One of skill in the art based on the benefit of this disclosure willreadily be able to identify acids having a pKa of within the aboveranges using reference materials available to one of skill in the artsuch as tables listing acids and their pKa values.

One example of a polysaccharide useful in the present invention is watersoluble chitosan, According to one embodiment, a polysaccharide usefulin the present invention includes chitosan having a degree ofdeacetylation between about 40% and about 99%, a percent of amine-basedsalt moieties between about 0% and about 100% and molecular weight rangebetween about 50 kDa and 2000 kDa or any range or value within thatrange whether overlapping or not as described above. The adhesivematerials can also be chitosan derivatives such as carboxymethylchitosan, hydroxyl propyl chitosan and the like or salts of suchderivatives such as salts of carboxymethyl chitosan, hydroxyl propylchitosan and the like.

According to certain exemplary embodiments, chitosan within the scope ofthe present invention can have a degree of deacetylation between about40% and about 99%, between about 40% and about 93%, between about 40%and about 75%, between about 45% and about 80%, between about 50% andabout 70%, between about 55% and about 75%, between about 50% and about65%, or between about 50% and about 60% or within any range or valuewithin the above ranges whether overlapping or not. According to oneembodiment, the degree of deacetylation is less than about 65%, lessthan about 60%, or less than about 55%.

According to an additional aspect of the present invention, the chitosanis yellow in color and thereby provides a natural color indicator of theside of the self adherent matrix to contact tissue and adhere. It is tobe understood that coloring ingredients, such as blue dye, could beadded to either the matrix or adhesive material to further distinguishthe side of the self adherent matrix to contact tissue and adhere. Inthis manner, a method is provided to identify an adhesive portion of anadherent resorbable matrix intended to contact a tissue site byutilizing color to distinguish the adhesive portion from the matrixportion. In this manner, the adhesive portion is identified by aparticular color, which may be the natural color of the adhesive orwhich may be due to the addition of a coloring agent such as a dye andan individual applying the adherent resorbable matrix will contact theadhesive portion having the particular color to intended tissue. In acertain embodiment, the matrix portion is identified by a particularcolor which may be the natural color of the matrix or which may be dueto the addition of a coloring agent such as a dye. Each of the adhesiveportion and the matrix portion may include a coloring agent to alter thecolor from the natural color of the adhesive portion and the matrixportion. According to an exemplary embodiment, the color of the adhesiveportion is different from the color of the matrix portion, so as todistinguish each portion. A coloring agent may be added to the adhesiveportion and/or the matrix portion in whole or in part. For example, theentire adhesive portion may be the same color such as when a colorant isadded during the manufacturing process and mixed homogeneously into theadhesive formulation prior to applying the adhesive formulation to thematrix. Similarly, the entire matrix portion may be the same color suchas when a colorant is added during the manufacturing process and mixedhomogeneously into the matrix formulation prior to lyophilization tofirm the solid matrix. According to alternate embodiments, only certainportions of the adhesive portion and/or the matrix portion may becolored. For, example, a colored pattern may be applied to the surfaceof either the adhesive portion or the matrix portion in any desiredpattern such as dots, stripes, etc. Any printing technique known tothose of skill in the art, such as application by hand or spray, silkscreen printing, stamping, ink jet, etc. is useful in the presentinvention. Similarly, a coloring agent may be used to print images orwords onto the surface of either the adhesive portion or the matrixportion, such as “Up,” “This Side Up,” “Apply This Side Away From Dura,”“Down,” “This Side Down,” or “Apply This Side To Dura.” Coloring agentsmay be any coloring agents known to those of skill in the art asindicated safe for human or animal consumption or safe for introductioninto a human or an animal. One of skill would readily identify suchcoloring agents from publicly available reference materials. Coloringagents may have any color such as red, orange, yellow, green, blue,indigo, violet or any combinations or shades or hues of these colors.Accordingly, one aspect of the present invention is directed to anadhesive resorbable matrix including a visible indicator to distinguishbetween the adhesive portion and the matrix portion. Such an adhesiveresorbable matrix is useful in a method of applying the matrix to tissuedescribed herein including the additional step of confirming throughvisual recognition the visible indicator and placing the portion of theadherent resorbable matrix with the visual indicator in contact with oraway from tissue.

According to certain aspects, one or more amine base units of chitosanare reacted with an acid to form a salt moiety. In this manner, thechitosan having salt moieties may be considered a chitosan salt. In asimilar manner, the chitosan has a salt content insofar as it includessalt moieties. According to certain other aspects, one or more aminebase units of chitosan are reacted to form a salt moiety with an acidhaving a pKa of between about 1.5 and 8.5, or with an acid having a pKaof between about 2.5 and 6.5, or with an acid having a pKa of betweenabout 4.0 and 6.0. One of skill in the art will recognize that theamount of salt moieties is dependent upon the pKa of the acid andchitosan along with the final pH of the solution. Salt substitution canchange simply by changing the pH of the solution. Similarly, basemoieties of other adhesive materials within the scope of the presentinvention can similarly be reacted with an acid to form salt moieties. Aparticular exemplary acid used to form a salt with one or more aminegroups of chitosan is acetic acid which has a pKa of about 4.7.According to one aspect, one or more acids with one or more differentpKa values can be reacted with the amine groups of chitosan to formvarious different salt moieties. For example, a mixture of acetic acidand lactic acid can be used to react with amine groups of chitosan toform different salt moieties on the chitosan. One of skill in the artbased on the benefit of this disclosure will readily be able to identifyacids having a pKa of between about 1.5 and 8.5, a pKa of between about2.5 and 6.5 or a pKa of between about 4.0 and 6.0 using referencematerials available to one of skill in the art such as tables listingacids and their pKa values. Such exemplary tables of dissociationconstants and pKa of organic acids in aqueous solutions and inorganicacids in aqueous solutions are found in the CRC Handbook of Chemistryand Physics 55^(th) Edition (1974-1975) at D-129 to D-130 herebyincorporated by reference. Other reference materials are known to existto those of skill in the art to identify pKa values for acids.

In addition, one of skill in the art will understand that the percent ofsalt moieties in chitosan may depend upon pH. Without wishing to bebound by theory, for example, for chitosan, at a pH of about 8 to about8.5, the ratio of salt to free base is about 0.03 to 1. When at a pH ofabout 5, the ratio of salt to free base is about 30 to 1. At a pH ofabout 6.5, the ratio of salt to free base is about 1 to 1. Accordingly,the percent of salt moieties created by acid reaction to total aminegroups before acid reaction ranges from about 0% (in the case of all orsubstantially all of the amines being in the free base form) to about100% (in the case of all or substantially all of the amines beingreacted to salt groups). The term chitosan free base can include somedegree of neutralization of acid with the amine groups to yield saltgroups, forming a water soluble or partially water soluble compound. Theterm chitosan salt can include some degree of amine groups present onthe chitosan. In additional exemplary embodiments, the percent of saltmoieties can range from between about 1% to about 99%, about 3% to about99%, about 10% to about 90%, about 10% to about 80%, about 20% to about80%, about 30% to about 70%, from about 40% to about 60%, from about 45%to about 55%, about 50% (i.e., a ratio of salt to amine of about 1 to 1)and any ranges and value in between whether overlapping or not. One ofskill in the art will understand that acids may be used that aresufficient to make a salt and a resulting water soluble or partly watersoluble polymer salt with any sufficient percent of salt moietiesconverted from base moieties.

According to certain embodiments, the chitosan free base has asolubility in water at 25° C. ranging from 0.01% to 99.9% dependent atleast in part on viscosity and molecular weight, for example, asolubility of about 1 to about 25%. According to certain embodiments,the chitosan free base has a purity defined for medical applications asless than about 100 EU/g. For example, it can have endotoxin levels lessthan about 40 EU/g. According to certain embodiments, the chitosan freebase can have a resorption time after positioning and hemostasis ofabout 1 to about 60 days in the human body, for example, a resorptiontime of about 3 to about 30 days, followed by resorption of the porousmatrix between about 0.5 days to about 2 years, for example about 30days to about 180 days, as tissue is regenerated. The resorption iscontrolled by the molecular weight of the free base or salt inconjunction with the composition and extent of crosslinking of thematrix.

According to certain embodiments, the chitosan salt or chitosanderivative salt can be organic or inorganic acid salts of chitosan orchitosan derivatives, for example, inorganic salts from the group ofhalides (F, Cl, Br, I), organic salts from amino acids or low MW organicacids, and diacids with these materials consisting of up to 12 carbonatoms. Examples include, but are not limited to, chitosan chloride,chitosan glutamate, chitosan ascorbate, chitosan lactate, chitosanmalate, chitosan acetate, and other pharmaceutically acceptable salts ofchitosan or chitosan derivatives. According to one embodiment, thechitosan salt is a hydrogen chloride, glutamic acid or a lactic acidsalt.

In further detailed aspects, the biocompatible adhesive can be acellulose, such as a modified cellulose including, for example,hydroxypropyl methyl cellulose. According to an additional embodiment, abiocompatible adhesive material includes a nonionic polymericpolysaccharide (including one of hydroxyethyl cellulose andcarboxymethyl cellulose), a synthetic nonionic polymer (including one ofpolyvinyl pyrrolidone, polyisobutylene, polyethylene glycol dimethylether, polysuccinimide, reverse phase matrix, poly (ethylene oxide), andpolyvinyl alcohol hydrolized), a nonionic fatty compound (including oneof alpha-tocopherol), a polyanionic polysaccharide (including one ofhyaluronic acid, chondroitin sulfate, oxidized dextran, oxidized starch,chondroitin sulfate N-hydroxysuccinimidyl ester, carageenan, cellulosecyanoethylated, xantham gum, and guar gum), a synthetic polyanionicpolymer including polyacrylic acids and polyacrylic acid polymers, apolycationic polysaccharide (including one of chitosan oligosaccharidelactate), a synthetic polycationic polymer (including one ofpolydiallyldimethyl ammonium chloride, poly (acrylimide-co-diallymethylammonium chloride), polyethyleneimine, and polycarbodiimide), apolycationic peptide (including one of albumin and poly 1-lysinehydrobromide), a salt (including one of calcium chloride and sodiumtetraborate), or a protein (including one of gelatin, thrombin, andfibrinogen) and combinations and modifications thereof.

A bioactive agent may be included in the adherent resorbable matrix,such as in either the matrix or the adhesive material or both. Thebioactive agent may include, for example, a neurotransmitter, a hormone,an immunomodulator, an immunosuppressant, an antibiotic, a cytostatic, adirurectic, a gastrointestinal agent, a cardiovascular agent, aneuropharamaceutical, a blood coagulation inducing agent, and the likeor a combination thereof. The matrix or adhesive preferably includeseffective amounts of meningeal tissue growth factors and/or bioactivepeptides, such as, e.g., RGD containing peptides, decorin, laminin,merosin, chondroitin sulfate, dermatin sulfate, heparin (bFGF),fibronectin and other integrin ligands, entactin, tenascin and the like.In certain embodiments, an effective amount of such an additive is about1 ug/mg matrix material such as 1 ug/mg collagen.

The bioactive agent may also include, for example, any drug, metabolite,or prodrug thereof, organic compound, substance, nutrient orbiologically beneficial agent including proteins, peptides (includingpolypeptides and oligopeptides), hormones, vaccines, oligonucleotides,genes, nucleic acids, steroids, antibiotics, antibodies, viruses, livecells, and other chemotherapeutic or non-therapeutic agents, or acombination thereof.

Suitable bioactive agents may also include, for example, a regenerativeagent such as one or more human growth factor-b, fibroblast growthfactor or vascular endothelial growth factor; or the agent may be a genetherapy agent, a cogener of platelet derived growth factors; or amonoclonal antibody directed against growth factors; or the agent may bea drug, a cell regeneration factor, drug-producing cells, orregenerative cells.

Due to the abundance of cationic amino groups along the structure ofchitosan, it is known that the drugs with carboxyl groups may beconjugated thereto and sustained release can be achieved through thehydrolysis of the amide or ester bonds linking drugs to the chitosanmolecule. As a polyelectrolyte, chitosan can electrostatically conjugatesensitive bioactive agents (e.g., recombinant proteins, such as VEGF)while preserving their bioactivities and enhancing their stabilities.

Suitable bioactive agents may further include, for example, progenitorcells of the same type of those from the vascular site, for example, ananeurysm, and progenitor cells that are historically different fromthose of the vascular site such as embryogenic or adult stem cells,which can act to stabilize the vasculature and/or accelerate the healingprocess. These cells may be incorporated into the matrix, the adhesive,or both the matrix and the adhesive.

Additional bioactive agents may include, for example, one or moresupplements, such as growth factors, polyclonal and monoclonalantibodies, and other compounds. Illustrative examples of suchsupplements include, for example, the following: fibrinolysisinhibitors, such as aprotonin, tranexamic acid and e-amino-caproic acid;antibiotics, such as tetracycline and ciprofloxacin, amoxicillin,protein C, heparin, prostacyclins, prostaglandins (particularly (PG1),leukotrienes, antithrombin III, ADPase, and plasminogen activator,steroids, such as dexamethasone, inhibitors of proptacyclin,prostaglandins, leukotrienes and/or kinins to inhibit inflammation;cardiovascular drugs, such as calcium channel blockers, vasodilators andvasoconstrictors; chemo attractants; local anesthetics such asbupivacaine; and antiproliferative/antitumor drugs such as5-fluorouracil, taxol and/or taxotere; antivirals, such as gangcyclovir,zidovudine, amantidine, vidarabine, ribaravin, trifluridine, acyclovir,dideoxyuridine and antibodies to viral components or gene products;cytokines, such as alpha- or beta- or gamma-Interferon, alpha- orbeta-tumor necrosis factor, and interleukins; colony stimulatingfactors, erythropoietin, antifungals, such as diflucan, ketaconizole andnystain; antiparasitic agents, such as pentamidine; anti-inflammatoryagents, such as alpha-1-anti-trypsin and alpha-1 antichymotrypsin;anesthetics, such as bupivacaine; analgesics; antiseptics; hormones;vitamins and other nutritional supplements; glycoproteins; fibronectin;peptides and proteins, carbohydrates (both simple and and/or complex);proteoglycans; antiangiogenins; antigens; lipids or liposomes;oligonucleotides (sense and/or antisense DNA and/or RNA); and genetherapy reagents.

The amount of bioactive agent incorporated into either the matrix oradhesive material or both depends upon the desired release profile, theconcentration of bioactive agent required for a biological effect, andthe length of time that the bioactive agent has to be released fortreatment, and should be within the discretion and wisdom of thepatient's attending physician. There is no upper limit on the amount ofbioactive agent incorporated into the biocompatible adherent sheet. Thelower limit of bioactive agent incorporated into the biocompatibleadherent sheet is dependent upon the activity of the bioactive agent,and the length of time needed for treatment. Specifically, in oneembodiment, the biocompatible adherent sheet can be formulated toprovide a one month release of bioactive agent. Alternatively, inanother embodiment, the biocompatible sheet can be formulated to providea three month delivery of bioactive agent. The biocompatible adherentsheet should release the bioactive agent contained within thebiocompatible adherent sheet at a controlled rate until thebiocompatible adherent sheet is effectively depleted of bioactive agent.

The adherent resorbable matrix provides a highly porous matrix for theinfiltration of cells (i.e. fibroblasts) and a substrate for thedeposition of new collagen upon implantation. The porous structure ofthe matrix facilitates the ingrowth of cells (i.e. fibroblasts) into theregenerative matrix. The device is gradually resorbed and replaced byendogenous connective tissue. The resorption depends on factors such asthe molecular weight of the cationic polysaccharide free base or saltand the composition and extent of crosslinking of the matrix.

The self adhesive resorbable matrix can be used for neurosurgical andorthopedic applications as a dural substitute for the repair andrestoration of dural defects in cranial and spinal surgical procedures.It can also be used in neurosurgical and orthopedic applications toclose dural defects following traumatic injury, excision, retraction, orshrinkage, or as a supplement to primary closure in cranial and spinalsurgical procedures. Other applications include, but are not limited to,wound, hernia repair, breast reconstruction, adhesion prevention,tendon/vessel protection, and other soft tissue repair. The adherentresorbable matrix may be used to close soft tissue defects from diabeticulcers, tunneling wounds, burns, traumatic injury or surgery and mayfunction as primary closure or an adjunct to primary closure.

According to certain embodiments, a thin layer of a chitosan free baseand/or chitosan salt such as an inorganic chitosan salt (such as achitosan chloride, chitosan bromide, chitosan iodide or chitosanfluoride salt) or an organic chitosan salt (including one of chitosanlactate, chitosan acrylate, chitosan malate, chitosan acetate orchitosan glutamate), between about 1 and about 60% of the total mass ofthe adherent resorbable matrix and with a molecular weight of about 10to about 2000 KDa, for example about 50 to about 150 kDa, is evenlyspread on one side of the highly biocompatible, porous, high puritymatrix. Chitosan free base, inorganic chitosan salts or organic chitosansalts and mixed chitosan salts are commercially available from AdjuvantPharmaceuticals (Adjuvant), Alpharetta, Ga. Chitosan chloride isavailable from FMC BioPolymer (Ewing, N.J.) or Carbomer (CA). Thechitosan free base, inorganic chitosan salt or organic chitosan salt isreconstituted to an about 0.1 to about 50 weight percent solution bysolubilizing in deionized or other purified water. The solution isapplied to the matrix using known methods and techniques.

The self adhesive resorbable collagen matrix can be cut into the desiredshape for use in the surgical site and can readily conform to thesurface of the underlying tissues. The self adhesive resorbable collagenmatrix may be used to close or seal soft tissue defects followingtraumatic injury, excision, retraction or shrinkage and may also be usedto supplement primary closure. The self adhesive resorbable collagenmatrix maintains its position in a wet field upon exposure to blood, CSFand irrigation. According to embodiments of the present invention, theadherent resorbable matrix is used to repair two physicallynoncontiguous tissues or portions thereof that are to be joinedtogether, or where a hole, tear, cut, perforation, or otherdiscontinuity is repaired so as to close the hole, tear, cut orperforation. The adherent resorbable matrix has at least some degree ofadhesion to the tissue to which the biocompatible adherent sheet isapplied, such that the sealed tissue is secured against at least amoderate displacing force. The discontinuity in the tissue that is beingsealed may be an incision made as part of a surgical procedure, or itmay be a wound.

In certain embodiments, the adherent resorbable matrix is indicated forapplication to a moist environment with limited liquid or body fluids.In one embodiment, application of the adherent resorbable matrixrequires gentle pressure for approximately 10 seconds. The tackiness ofthe adhesive layer when exposed to a moist environment is characterizedby an increase in uniaxial tensile adhesion (using for example ASTMF-2258-05) from about 0.4N to about 10N as compared to less than 0.4 Nfor a matrix without an adhesive layer. In simulated application to avertical surface of protein rich substance (sausage casing, Nippi Inc.,Tokyo, Japan), the adherent resorbable matrix of the present inventionexhibited a resistance to migration for up to 180 seconds under acontinuous flow of water or saline at 30-40 ml/s. In comparison, theaverage resistance to migration of a single layer collagen matrixwithout an adhesive is less than one second.

According to other embodiments, the adherent resorbable matrix issuitable for repairing intentional damage to the meningeal tissues, asin surgery, and consequential damage to the meningeal tissues, as mightoccur as a result of accidental head trauma. For example, after brainsurgery, the adherent resorbable matrix of the present invention isinserted to occupy space left by the removal resultant on surgery. As tomeningeal repair following a craniotomy or a laminectomy, particularlywith the incision through the dura, the adherent resorbable matrix ofthe present invention can simply be implanted on contact with thecranial or spinal dural defect created by the surgery. Although it canbe preferred to simply contact the damaged meningeal tissue and adjacentundamaged tissue with the adherent resorbable matrix (particularly whenthe adherent resorbable matrix is being used as a cranial durasubstitute), the product can also be mechanically bonded (e.g., sutured)and/or chemically bonded to the damaged tissue and adjacent undamagedtissue (e.g., fibrin glue). The adherent resorbable matrix preferablyconnects undamaged portions of meningeal tissue adjacent to the damagedmeningeal tissue by overlapping these undamaged tissues. The damagedtissue can be, e.g., torn, cut, excised or lacerated, and can be locatedin e.g., the human spinal dura or the human cerebral dura. Regeneratedmeningeal tissue grows within the product, while the product remainsimplanted within a patient. That is, the product acts as a matrix orscaffold for tissue growth, such as for reparative tissue growth.According to certain embodiments, the product is sustainably resorbedwithin about three months after implantation. Although the product ofthe invention is particularly suitable for dural repair, it is alsosuitable for promoting tissue growth and/or wound healing in othercontexts. For example, the product is suitable for use as abioresorbable pledget to assist in suturing, a suturable hemostaticdevice, hernia patches, pericardial patches, and the like.

Accordingly, the adherent resorbable matrices of the present inventionhave neurosurgical and orthopedic applications as a dural substitute forthe repair and restoration of dural defects in cranial and spinalsurgical procedures. The adherent resorbable matrices of the presentinvention have neurosurgical and orthopedic applications to close duraldefects following traumatic injury, excision, retraction or shrinkage.The adherent resorbable matrices of the present invention haveneurosurgical and orthopedic applications as a supplement to primaryclosure in cranial and spinal surgical procedures. The adherentresorbable matrices of the present invention have applications in woundrepair, hernia repair, breast reconstruction, tissue adhesionprevention, tendon and/or vessel protection and other soft tissuerepair.

The following examples are specific embodiments of the present inventionbut are not intended to limit it.

Example 1 Preparation of an Exemplary Matrix

A matrix according to the present invention including a bioresorbablenatural, semisynthetic or synthetic polymer or a combination thereof isprepared according to the following method using bovine collagen as anexemplary polymer to form a matrix having sponge-like characteristics.The method includes steps that are recognized as effective forinactivating viral and prion contamination, thereby increasing safetyand reducing inflammatory response. According to one embodiment, thematrix is substantially free of viruses and prions without beingphysiologically incompatible. The phrase “substantially free of virusesand prions” means that the product does not contain infection-effectiveamounts of viruses and prions. In one aspect, collagen is treated by aprocess sufficient to achieve at least a 4 log clearance of virus, or atleast a 6 log clearance of virus, or at least an 8 log clearance ofvirus, as measured with a statistical confidence level of at least 95%.For example, if the concentration of virus before treatment is 10⁷ andafter treatment is 10¹, then there has been a 6 log clearance of virus.

In general, a self adhesive resorbable collagen matrix can be preparedby providing a collagen dispersion slurry; lyophilizing the collagenslurry until moisture is removed to produce a collagen sheet (or otherdesired shape or form); cross-linking the lyophilized collagen sheets;making an aqueous solution of an adhesive; applying the adhesivesolution onto the lyophilized and cross-linked collagen sheets; andlyophilizing the sheet to produce the self adhesive resorbable collagenmatrix. The self adhesive resorbable collagen sheet can be cut into thedesired size and shape, packaged, and sterilized for commercialization.

More particularly, the adhesive matrix can be prepared by applying athin layer of an inorganic or organic salt or a water soluble orpartially water soluble free base or some combination thereof of thecationic (chitosan) polysaccharide adhesive (between 1 and 60% of thetotal mass of the device) evenly or in an intermittent pattern over oneor more sides of the porous collagen matrix. The cationic polysaccharidefree base or salt component is reconstituted to an about 1 to about 40weight percent solution by solubilizing the sheet, powder or granules indeionized or other purified water or polar protic solvent or polaraprotic solvent or some combination thereof. The adhesive is applied asa mixture, slurry or solution to the collagen matrix manually using aspatula, draw-down bar, spraying, or similar method directly onto thematrix or the adhesive material may be applied to a rigid surface andsubsequently transferred to the porous matrix. An adhesive solution mayalso be applied using a patterned grid over the surface of the matrixand spreading the adhesive solution over the grid, delivering thesolution to a specific percentage of the surface area with a knownvolume delivered. In the liquid form the adhesive is allowed topartially absorb into the surface of the porous matrix through capillaryaction. The extent of the adhesive penetration into the matrix isthermally controlled whereas the construct is rapidly frozen at specifictime points to arrest the process. The device is then lyophilized usingstandard methods for a minimum of 6 hours or until completely dried. Thedry device is cut, packaged in double blister or foil packaging, andexposed to ethylene oxide gas, electron beam, or gamma irradiation forsterilization.

In accordance with the present invention, a collagen dispersion isprepared according to methods well known in the art. A native source ofType I collagen, such as skin, tendons, ligaments or bone, is firstmechanically or hand cleaned of fat, fascia and other extraneous matterand washed. The cleaned and washed collagen containing material is thencomminuted, generally by slicing or grinding. The material is thensubjected to an enzyme treatment while under intermittent stirring witha proteolytic enzyme, such as ficin, pepsin, and the like, so as toremove non-collagenous impurities which may cause antigenic activity andto swell the collagen by removing elastin. The amount of enzyme added tothe collagen material and the conditions under which enzyme digestiontakes place is dependent upon the particular enzyme being used.Generally, when using ficin, which is most commonly used, the pH isadjusted to about 6.0 to 6.3, and the collagen material is digested forabout 1 to 2 hours at a temperature of about 36.5° C. to 37.5° C. withone part ficin for every 150 parts of collagen material. After therequisite amount of time, the enzyme is inactivated by appropriate meanswell known in the art, such as by the addition of a solution of anoxidizing agent, such as sodium chlorite when the enzyme is ficin. Theenzyme treated collagen containing material is then washed to removeexcess enzyme and the non-collagenous protein impurities. Preferably,the washing is carried out with ultrafiltered and deionized water andoptionally further washed with dilute aqueous hydrogen peroxide.

The enzyme digested collagen containing material is then furthersubjected to an alkali treatment at a pH of about 13 to 14, at atemperature of about 25° C. to 30° C. for a period of about 35 to about48 hours. An exemplary period is about 40 hours. The alkali treatment iscarried out in an aqueous solution of 5% sodium hydroxide and 20% sodiumsulfate. This alkali treatment removes contaminating glycoproteins andlipids. The solution is then neutralized with a suitable acid, such asaqueous sulfuric acid, and thoroughly washed. The collagen material isthen further swollen with a suitable acid solution which acid does notcause any cross-linking of the collagen. Such acids are well known tothose skilled in the art and include acetic acid, hydrochloric acid,lactic acid, and the like. Regardless of which acid is used, the pH ofthe acid collagen dispersion is in the range of about 2 to 3.

The dispersed collagen mixture is then homogenized by any conventionalmeans, such as a blender or homogenizer, so as to further dissociate thefibers and is then filtered to remove unswollen, non-collagenousmaterial by means well known in the art, such as by passing thedispersion through a 100 mesh stainless steel screen. The resultingfiltered collagen dispersion can then be used to prepare the matrix ofthe present invention.

The matrix can be prepared by lyophilization of a collagen dispersionprepared according to the patent, preferably having a concentration ofbetween 0.1 and 10% solids (w:w) and more preferably at least 1.0%solids. A volume of the dispersion is poured into a suitable (preferablynon-stick) tray to provide a matrix having a suitable shape. The matrixhas a thickness from about 2.5 mm to about 5 mm. An exemplary thicknessis about 3 mm. The dispersion is then frozen and lyophilized for about 1to about 48 hours. The density of the dispersion and the lyophilizationcycle affect the matrix density and pore, size. The matrix density isabout 0.0001 mg/mm³ to about 0.12 mg/mm³. An exemplary density is about0.009 mg/mm³.

The matrix of the present invention has sponge-like properties and canbe referred to as a sponge. The matrix of the present invention includespores of a sufficient size and quantity to permit growing meningealtissue to infiltrate therein. The pore size ranges from about 10 μm toabout 500 μm, and about 50 μm to about 150 μm, with surface pores beingsmaller than cross-sectional (internal) pores. In certain exemplaryembodiments, the surface pores range in diameter from about 30 μm toabout 150 μm. An exemplary surface pore size is about 70 μm. Thecross-sectional pores range in diameter from about 50 μm to about 300μm. An exemplary cross-sectional pore size is about 70 μm.

Example 2 Preparation of an Adhesive Solution and Application to aMatrix

The adhesive can be applied in any desired pattern on one or moresurfaces of the matrix that are to contact tissue. One of skill in theart, given the geometry of the matrix and its intended application, willunderstand where adhesive is to be applied given the portion of thematrix that is to contact tissue. For example, if the matrix is in theform of a sheet, the adhesive material can be applied to one or bothsurfaces given the desired utility of the sheet. According to certainaspects, the adhesive layer can have a thickness of between about 0.10mm to about 1 mm, about 0.12 mm to about 0.8 mm, about 0.18 to about 0.6mm, from a bout 0.2 mm to about 0.5 mm, from about 0.3 mm to about 0.4mm and any ranges or value within the above ranges whether overlappingor not. One of skill will recognized based on the present disclosurethat the thickness of the adhesive layer can depend on the particularadhesive formulation and the desired adhesive property.

According to one aspect, a thin layer of the adhesive (between 10 and40% of the total mass of the device) is evenly spread onto one side of alyophilized collagen sponge. Adhesive materials within the scope of thepresent invention, such as chitosan having a molecular weight of about75 kDa to about 600 kDa and a degree of deacetylation of about 50% toabout 55% and chitosan chloride salt are commercially available fromsources such as Adjuvant and FMC respectively and are reconstituted toan about 1 to about 50 weight percent solution, about 3 to about 40weight percent solution, about 5 to about 20 weight percent solution andany ranges or values therein whether overlapping or not, by solubilizingthe adhesive material in deionized or other purified water. The solutionis applied to the collagen matrix by an appropriate method known in theart, for example, by hand using a spatula or drawdown bar method.Partial absorption of the solution including the adhesive material intothe top layer of the collagen sponge is thermally controlled. It is tobe understood that higher weight percent adhesive material solutions aregenerally more viscous and thus tend to absorb less into the surface ofthe matrix material. Higher weight percent adhesive material solutionsalso create a more dense adhesive layer when lyophilized. The device isthen lyophilized (Virtis or other lyophilizer) for a period of time suchas 12 hours or until completely dried. The dry device is cut, packagedin double blister packaging, and exposed to ethylene oxide gas forsterilization.

Chitosan materials having acetate salt groups used in the examples werepurchased from Adjuvant Pharmaceuticals L.L.C., Marietta, Ga. Thechitosan materials in the examples are indicated as being LMW (lowmolecular weight), MMW (medium molecular weight) or HMW (high molecularweight.) Chitosan materials designated as LMW were obtained fromAdjuvant LOT #109-002 and were characterized as follows: Appearance: offwhite powder; Viscosity as measured by a Brookfield Viscometer in a 1%solution in 1% acetic acid: 4 CPS; Weight Loss on Drying 1 g at 60° C.in vacuum oven, ON: 2%; pH in a 1% aqueous solution: 5.3; GPC MolecularWeight: 100 kDa; Degree of Deacetylation as determined by NMR: 55%; andProtein Content: 0.04%. Chitosan materials designated as MMW wereobtained from Adjuvant LOT #88-56 and were characterized as follows:Appearance: off white powder; Viscosity as measured by a BrookfieldViscometer in a 1% solution in 1% acetic acid: 45 CPS; Weight Loss onDrying 1 g at 60° C. in vacuum oven, ON: 3%; pH in a 1% aqueoussolution: 5.0; GPC Molecular Weight: 696.3 kDa; Degree of Deacetylationas determined by NMR: 60%. Chitosan materials designated as HMW wereobtained from Adjuvant LOT #109-018 and were characterized as follows:Appearance: off white powder; Viscosity as measured by a BrookfieldViscometer in a 1% solution in 1% acetic acid: 12.8 CPS; Weight Loss onDrying 1 g at 60° C. in vacuum oven, ON: 5%; pH in a 1% aqueoussolution: 5.5; GPC Molecular Weight: 1000 kDa; Degree of Deacetylationas determined by NMR: 50%; Endotoxins: 9 EU/gram and Protein Content:0.04%.

A exemplary method to create a viscous, aqueous solution fromcommercially available chitosan powder and to coat a collagen matrixwith the aqueous solution is as follows. Chitosan is a water-solublepowder and is a solid at room temperature in its pure state. Chitosancan be dissolved at a known concentration in WFI product water to createan aqueous solution of chitosan. A 1% solution of chitosan is preparedand the viscosity determined. Exemplary amounts of chitosan added toproduce a desired weight percent solution are presented in Table 1below.

TABLE 1 1 wt % Viscosity 3-6.99 cps 7-12.99 cps 13-19.99 cps 20-26.99cps 27-50 cps Viscosity of 18 wt % 15 wt % 12 wt % 9 wt % 6.5 wt %solution Chitosan to add 0.18 grams 0.14 grams 0.10 grams 0.09 grams0.065 grams per 1 ml water (C)

Determine a desired volume of solution (B). Place 250 ml beaker on astir plate. Using a pipette gun and 50 ml pipette, fill the beaker withthe volume of WFI product water determined above (B). Place stir bar inwater and stir at 300 rpm. Using the determined viscosity, determinechitosan mass value (C) from Table above, and measure out appropriatemass of chitosan to prepare a 6.5-18 wt % solution as determined below.The mass of chitosan in solution (D) is determined by multiplying B byC. Add chitosan slowly to stirring water, allowing time to dissolve. Asnecessary, stir by hand with spatula and/or adjust stir bar rotation toachieve even stirring. Weight percent solution created is equal toD/B*100%. Continue stirring until no dense powder clumps can bevisualized. Place solution in 2-8° C., covered with parafilm untilneeded.

An 18 wt % aqueous chitosan solution is applied to a collagen spongematrix as follows. For purposes of creating a thin (0.3 mm)water-soluble coating, chitosan can be dissolved at a knownconcentration in deionized water and spread evenly across 22 in×20in×3.5 mm dry and vapor crosslinked sheets of collagen matrix made of1.0% solids alkali dispersion. In manual mode, initiate freeze mode on alyophilizer with temperature set point set to −40° C. Allow at least 1hour to elapse before continuing to ensure that the lyophilizer is atthe set temperature before loading. Place 8 in×10 in HDPE sheet on hard,flat surface. Align 300 micron draw down bar at the end of the rigidsurface secured by the 1 kg mass. Cut sheets of in process alkali sheetsof collagen sponge into equal quarters, yielding four (4) equal sizedsponges, approximately 8 in×10 in. Record the “pre-mass” of each sheetin a log. Remove chitosan solution from 2-8° C. storage and allowseveral hours to come to room temperature. Pour approximately 25 ml ofchitosan solution from its container and spread on the plastic sheet ina line in front of the draw down bar. Grasp the 300 micron drawn bar byboth handles and draw down the solubilized chitosan in a thin filmacross the plastic sheet. Allow at least 5 seconds for the material toself-level. Place the smooth side of the collagen alkali sheet againstthe thin film of chitosan. Place a 10 in×12 in stainless steel mesh ontop of the sample for gentle, even pressure against the layer ofchitosan. Wait 2 minutes and remove the 10 in×12 in mesh. Transfer the10 in×12 in tray with sample immediately to a lyophilizer with shelvespre-frozen to −40° C. freezer. The sample should contact the lyophilizer2-2.5 minutes after first contact of sponge to chitosan layer. Once alllyophilizer shelves are loaded and the door secured, wait at least 20minutes to allow the sample loaded last to reach freezing temperature.Stop the manual shelf freezing and begin a 12 hour lyophilization cyclebeginning at −35° C. as follows:

FREEZING TEMP −35 −35 TIME 0 30 EXTRA FREEZE TEMP −35 TIME 1 PRESS 200PRIMARY DRYING TEMP −35 −20 −20 −15 −15 −5 −5 25 25 TIME 30 30 30 30 3030 60 150 300 PRESS 200 200 200 200 200 200 200 200 200

After 12 hours, remove samples from lyophilizer and record “post-mass”in a log. In the event that cutting and packaging cannot immediatelyfollow this step, store lyophilized sheets on HDPE in a dry, protectedcontainer (up to 1 week) until needed. Defrost and clean lyophilizer.

Example 3 Preparation of a Self Adhesive Resorbable Collagen Matrix witha Chitosan Salt Adhesive Layer

A 3.5 wt % chitosan chloride solution was prepared by adding 0.7 g ofchitosan chloride salt, (FMC BioPolymer) in incremental quantities of0.2 g to 20 ml of deionized (DI) water. The solution was stirred by handafter each incremental addition of chitosan chloride salt. Next, thesolution was split equally into two beakers. A 5.5 wt % solution wasthen prepared by adding 0.2 grams of chitosan chloride salt to onebeaker of the 3.5 wt % solution and stirring by hand. Both solutionswere transparent and homogenous containing no visible solids. Threemilliliters of each solution was then applied to a 3″×3″ collagensponge. Application was conducted by hand using a stainless steelspatula at a ratio of 0.33 ml per square inch. Each sample was left atroom temperature for three minutes, and then frozen and lyophilizedovernight to remove any excess water. The chitosan chloride left an offwhite/yellow solid on the treated side of the collagen sponge.

Additional matrix samples were prepared with chitosan glutamate (FMCBioPolymer) by utilizing the methodology described above. Samples wereprepared with a 5 wt % salt solution and a 7 wt % salt solution.

A 3.5 wt % solution was prepared by adding 1.5 g chitosan chloride salt(FMC BioPolymer) to 50 ml DI water. A 5.5 wt % solution was prepared byadding 1 g chitosan chloride salt (FMC BioPolymer) to 18 ml of DI water.A 15 wt % solution was prepared by adding about 3.0 g chitosan chloridesalt (FMC BioPolymer) to 20 ml of DI water. Each solution was drawn downby a draw-down bar (TMI Industries Bar No 300) on a plastic sheet andthe backing of each collagen sponge was placed on the solution. Thesamples were left at room temperature for a period of time and were thenfrozen and lyophilized to remove any residual water.

Additional matrix samples were prepared utilizing other chitosan salts(CarboMer, Inc.). The samples were prepared as described immediatelyabove. If solutions were not homogenous they were applied by hand ratherthan by draw-down bar. The salts utilized and their concentrations arepresented in Table 2.

TABLE 2 How applied to Time at room Salt Concentration collagen spongetemperature Chitosan glutamate 5 wt % Hand applied 3 minutes Chitosanglutamate 7 wt % Hand applied 3 minutes Chitosan lactate 5 wt % Drawdown applied 1 minute Chitosan lactate 8 wt % Draw down applied 1 minuteChitosan malate Hand applied 1 minute Chitosan ascorbate 5 wt % Handapplied 1 minute Chitosan ascorbate 7 wt % Hand applied 1 minute pH 3.5Chitosan ascorbate 7 wt % Hand applied 1 minute pH 1.5 Carboxymethyl 6wt % Hand applied 1 minute chitosan

Example 4 Tension Adhesion Testing of a Self Adhesive ResorbableCollagen Matrix

To determine the ability of various self adherent matrices to adhere totissue under aqueous conditions, a tension adhesion test was performed.The test was performed on matrix samples identified in the Table 3below. Some matrix samples were sterilized prior to testing.Sterilization occurred by either electronic beam (e-beam) sterilizationor ethylene oxide (EO) sterilization.

Initial set up for the adhesion test required preparing aluminum T barsto be used with an Instron machine (Model 5544). Loctite glue wasuniformly applied to an aluminum T bar. Next, a matrix sample was placeduntreated side up. The T bar was placed glue side down on the untreatedside of the sample and held in place for 20 seconds using two 0.7 Nweights. The weights were removed and the matrix sample was trimmedaround the T bar. While trimming the matrix sample it was verified thatthe matrix sample was completely attached to the T bar. The T bar withthe attached matrix was then set aside.

A second aluminum T bar was then prepared by uniformly applying Loctiteglue to the bar. The T bar was then placed, glue side down, onto freshlythawed bovine pericardium (Spears Biologics) and held in place for 40seconds with two 1.3 N weights. The weights were then removed and thepericardium was trimmed around the T bar. While trimming the pericardiumit was verified that it was completely attached to the bar. The preparedT bar was then inserted into the top grip of the Instron machine.

Prior to inserting the previously prepared T bar with the attachedmatrix into the Instron machine, 1.0 ml of Phosphate buffer saline (PBS)was injected into the matrix. Within 50 seconds of applying the PBS tothe matrix, the T bar was placed in the bottom Instron grip and the topand bottom grips were aligned to be parallel to one another andapproximately 1 mm apart. An adhesion-test program was then run. Themachine applied a preload of 0.7 N for 30 seconds to the matrix sampleand the pericardium substrate, which was followed by a quasi statictensile loading to evaluate the peak adhesive force obtained between thematrix and the substrate. The matrix samples tested and their averagetensile adhesive strength are shown in Table 3 which were considerablyabove the average tensile strength determined by the above protocol of acontrol collagen matrix that did not have an adhesive layer.

TABLE 3 Average Tensile Adhesive Sample Strength (N) 5.5 wt % ChitosanChloride (pre-sterile) 0.99074 3.5 wt % Chitosan Chloride (pre-sterile)0.74203   7 wt % Chitosan Glutamate (pre-sterile) 0.79   5 wt % ChitosanGlutamate (pre-sterile) 1.42 5.5 wt % Chitosan Chloride (e-beamsterilized) 1.04742 3.5 wt % Chitosan Chloride (e-beam sterilized)0.96424   7 wt % Chitosan Glutamate (e-beam sterilized) 1.00   5 wt %Chitosan Glutamate (e-beam sterilized) 1.22   5 wt % Chitosan Lactate(EO sterilized) 0.54577   8 wt % Chitosan Lactate (EO sterilized)3.07484   3 wt % Chitosan Chloride (EO sterilized) 0.50169  15 wt %Chitosan Chloride (EO sterilized) 0.92587 5.5 wt % Chitosan Chloride (EOsterilized) 0.60677   6 wt % Carboxymethyl Chitosan (EO sterilized)0.55489   7 wt % Chitosan Ascorbate pH 3.5 (EO sterilized) 0.64474   7wt % Chitosan Ascorbate pH 1.5 (EO sterilized) 1.22051   5 wt % ChitosanAscorbate pH 3.5 (EO sterilized) 0.77322  10 wt % Chitosan Malate (EOsterilized) 0.53632   5 wt % Chitosan Lactate (e-beam sterilized)1.00016   8 wt % Chitosan Lactate (e-beam sterilized) 1.00759   3 wt %Chitosan Chloride (e-beam sterilized) 1.88849  15 wt % Chitosan Chloride(e-beam sterilized) 1.30108 5.5 wt % Chitosan Chloride (e-beamsterilized) 1.02817   6 wt % Carboxymethyl Chitosan (e-beam sterilized)0.59525   7 wt % Chitosan Ascorbate pH 1.5 (e-beam sterilized) 0.63881

Additional chitosan samples were tested for adhesion using a collagenmatrix (DURAGEN XS) without adhesive as a control. The results arepresented in Table 4 below.

TABLE 4 TENSION load avg ADHESION TESTING sample (N) load (N DuraGen XScontrol 0.23 0.23 control 0.22 DuraGen XS control 0.62 0.43 control 0.24DuraGen XS control 0.41 0.36 control 0.31 DT-92 (70:30 LMW 1 1.40 1.03chltosan: MMW chltosan) 2 1.03 3 0.76 4 0.92 DT-92 (30:70 LMW 21 1.981.70 chltosan: MMW chftosan) 22 1.45 23 1.78 24 1.60 DT-93 (MMWchitosan) 1 1.46 1.30 2 1.15 3 1.22 4 1.37 DT-94 (18 wt % 4 1.25 1.78LMW chitosan) 26 1.24 53 3.45 71 1.47 88 1.48 144 2.19 2.47 146 2.89 1471.68 148 2.80 149 2.78 11 1.77 1.52 12 2.76 21 0.92 23 0.99 27 1.15DT-95 (70:30 LMW 4 2.87 2.18 chltosan: MMW chitosan) 11 1.47 24 1.99 292.64 42 1.94 64 2.31 3.30 66 2.97 67 3.27 68 4.15 69 3.81 DT-97 (30:70LMW 3 2.28 2.55 chitosan: MMW chltosan) 4 3.40 6 1.20 7 3.31 DT-98 (MMWchitosan) 4 3.96 3.28 5 2.96 6 2.43 7 3.77

Example 5 Vertical Slide Testing of a Self Adhesive Resorbable CollagenMatrix Capable of Adhering to Tissue Under Aqueous Conditions

To further verify the ability of various chitosan salts to adhere totissue under aqueous conditions, a vertical slide test was performed.The test was performed on matrix samples identified in Table 5 below.Initially, a 10″×12″ anodized aluminum tray was placed on a hot plate tobe heated to 37±3° C. The tray was marked at 5.5″ and 6.5″ from the 10″edge. A 1 L beaker was then inverted and placed in a large containmenttray and a 5 L carboy was placed on a 12″ stool. Tygon tubing was runfrom the pour spout of the carboy and attached to the beaker. The tubingoutlet was positioned downward at a 45° angle exactly 6.5″ above thetable surface.

Sample preparation was begun by cutting homogenized collagen sausagecasing (Nippi) into 2″ wide strips and adhering it to the preheatedaluminum tray using water surface tension. The sausage casing strip waswet with 0.25 ml DI water. A matrix sample was then placed salt treatedside down on the strip. A 62 g non-metallic mass was gently placed onthe sample for 10 seconds. After the weight was removed, 0.75 ml DIwater was directly applied to the sample. The tray with the sampleattached was then placed in a vertical position and the hose outlet wasplaced so it gently abutted the sample. The temperature of the sausagecasing was taken to verify it was 37±3° C. Next, the sample wasirrigated with water from the carboy. The irrigation occurred at a flowrate of 30 to 40 ml/sec. The time it took for the sample to fall fromthe tray or for 5.5 L to have irrigated was recorded. The matrix samplestested and their average irrigation time are displayed in Table 5.

TABLE 5 Average Sample Duration (sec) 5.5 wt % Chitosan Chloride (presterile) 69.63 3.5 wt % Chitosan Chloride (pre sterile) 106.62   7 wt %Chitosan Glutamate (pre sterile) 143.15   5 wt % Chitosan Glutamate (presterile) 160.04

Additional chitosan samples were tested for vertical slide using acollagen matrix (DURAGEN XS) without adhesive as a control. The resultsare presented in Table 5 below.

TABLE 5 VERTICAL time avg SLIDE TESTING sample (sec) time (sec) DuraGenXS Control 0.49 N/A Duragen XS Control 0.51 0.51 DuraGen XS Control 0.360.36 DT-92 (70:30 LMW chitosan: 1 85.62 55.39 MMW chitosan) 2 32.01 364.92 4 39.02 DT-92 (30:70 LMW 21 184.78 156.62 chitosan: MMW chitosan)22 165.89 23 177.93 24 97.88 DT-93 (MMW chitosan) 1 161.39 133.46 289.72 3 158.77 4 123.94 DT-94 (18 wt % 4 99.68 92.91 LMW chitosan) 2646.04 53 114.54 71 92.91 88 111.40 144 91.70 105.33 146 86.63 147 204.47148 68.34 149 75.53 11 86.88 71.84 12 108.35 21 39.90 23 62.83 27 61.24

Example 6 In Situ Testing of a Self Adhesive Resorbable Matrix Capableof Adhering to Tissue Under Aqueous Conditions

To quantitatively assess the adhesive bonding of a matrix in anenvironment similar to that encountered during surgery, a porcine modelwas utilized. The test was performed on various matrix samples.Initially, a porcine cranium was opened and the brain surface exposed toconfirm the dura matter was intact. Once the dura was exposed, a dry 2×2cm matrix sample was placed on an untested area of the dura that hadbeen wet with PBS. The sample was placed in a vertical position and heldin place for 10 seconds using a flat press. Alternatively, a sample wasplaced in a horizontal position and held in place for 10 seconds usingfingertips. The sample was then wet out prior to irrigation. Next, thesample was irrigated with increasing force until it migrated. If thesample did not migrate a peel test was performed to assess the adherenceof the sample to the dura. For some samples, additional tests wereperformed to assess the ability of the matrix samples to reattach to thedura after being removed, as well as their ability to attach to an areaof the dura that had previously been treated.

The matrix samples tested include: chitosan chloride e-beam sterilized;chitosan chloride EO sterilized; chitosan chloride pre-sterilization;chitosan glutamate e-beam sterilized; chitosan lactate e-beamsterilized; chitosan lactate EO sterilized; chitosan ascorbate e-beamsterilized; chitosan ascorbate EO sterilized; chitosan acetatepre-sterilization; and carboxymethyl chitosan e-beam sterilized.Chitosan salts provided adequate resistance to migration in a porcinecranium evaluation and significantly greater than the DURAGEN matrixproduct without adhesive.

Alternatively, a matrix sample was placed on an untested area of durathat had been wet with PBS. The sample was placed in a more extremevertical position and held in place for 10 seconds using fingertips. Thesample was then wet out and irrigated with increasing force. Afterirrigation a manual peel test was performed and the sample wasrepositioned and held in place for 30 seconds using fingertips. Thesample was then irrigated again until migration occurred.

Another alternative was to place a matrix sample on an untested area ofthe dura that had been wet with PBS. The sample was placed in a moreextreme vertical position and held in place for 10 seconds usingfingertips. After attachment and prior to wetting out, it was attemptedto manually slide the sample out of place. The sample was then wet outand a manual peel test was performed. The sample was then repositionedon new dura and irrigated until migration occurred.

A final test of sample adherence was to handle the matrix sample withmoist/wet gloves prior to attaching the sample to the dura as describedabove in an extreme vertical position. The sample was then wet out andirrigated with increasing force until migration occurred. If the sampledid not migrate a manual peel test was performed.

The experiments showed that the self adhesive resorbable matrix withchitosan chloride as the adhesive demonstrated consistently excellentresistance to migration when applied to dry to moist dura and wet outprior to irrigation.

Example 7 In Situ Testing of a Self Adhesive Resorbable Matrix Capableof Adhering to Tissue Under Aqueous Conditions

In addition, the following experiments were carried out using theporcine cranium model described above. The porcine model was utilized toobtain a semi-quantitative assessment of the functional performance ofmatrices having various adhesive layers of deacetylated chitosan (CHN)purchased from Adjuvant Pharmaceuticals, Alpharetta, Ga. Chitosanchloride salts were purchased from FMC BioPolymer, Ewing, N.J. Thechitosan tested had a degree of deacetylation of between 50% to 80%, amolecular weight between about 50 kDa and 1000 kDa and a salt content ofbetween 3% to 99%. The adhesive ability of the sample was graded on ascale of 1 to 10 with 1 being the lowest adhesive capability and 10being the highest adhesive capability. Generally, a 10 was assigned forresistance to 5 consecutive irrigations without any sign of lostadhesive properties upon peeling the sample loose from the dura. Themiddle numbers in the scale were a linear range between the twoextremes, for samples that resisted 1, 2, 3, or 4 irrigations butdisplayed some degree of decreased adhesive force. A control lacking anadhesive layer resulted in a “0 out of 10.” The results are presentedbelow.

An approximately three month old sample of 5.5 wt % chitosan chloride(FMC), 0.3 mm adhesive thickness, EO (DT-67-107) was placed on moistdura, which had been moistened with PBS. Sample was held in place for 10seconds using a single finger; the sample was wet out prior toirrigation. The sample was irrigated five times. The sample providedsignificant resistance to peel when lifted with forceps and wasevaluated as a “10 out of 10.”

A sample of 5.5 wt % chitosan chloride (FMC), 0.3 mm adhesive thickness,EO (DT-67-107) was placed on moist dura, which had been moistened withPBS. Sample was held in place for 10 seconds using a single finger; thesample was wet out prior to irrigation. The sample did not migrate after5 irrigations, and there was significant pull on the dura when thesample was peeled loose from the dura and was evaluated as a “10 out of10”.

A sample of 18 wt % LMW chitosan, 0.3 mm adhesive thickness, EO(DT-76-70) was placed on moist dura, which had been moistened with PBS.Sample was held in place for 10 seconds using a single finger; thesample was wet out prior to irrigation. The sample did not migrate after5 irrigations, and there was significant pull on the dura when thesample was peeled loose from the dura and was evaluated as a “10 out of10”.

A sample of 6.5 wt % MMW chitosan, 0.3 mm adhesive thickness, EO(DT-89-1) was placed on moist dura, which had been moistened with PBS.Sample was held in place for 10 seconds using a single finger; thesample was wet out prior to irrigation. The top corner of the samplelifted after 4 irrigations but did not migrate after 5 irrigations.There was minimal pull on the dura when the sample was peeled loose andwas evaluated as a “7 out of 10”.

A sample of 6.5 wt % HMW chitosan, 0.3 mm adhesive thickness, EO(DT-76-33) was placed on moist dura, which had been moistened with PBS.Sample was held in place for 10 seconds using a single finger; thesample was wet out prior to irrigation. The sample edge of the samplelifted after the first irrigation, and migrated after the secondirrigations and was evaluated as a “2 out of 10”.

A sample of 80:20 LMW:HMW chitosan, 0.3 mm adhesive thickness, EO(DT-83-2) was placed on moist dura, which had been moistened with PBS.Sample was held in place for 10 seconds using a single finger; thesample was wet out prior to irrigation. The sample did not migrate after5 irrigations. A peel test demonstrated significant pull against thedura and was evaluated as a “8 out of 10”.

A sample of 6 wt % chitosan chloride (FMC) with an edge defect, 0.3 mmadhesive thickness, EO (DT-67-107) was placed on moist dura, which hadbeen moistened with PBS. Sample was held in place for 10 seconds using asingle finger; the sample was wet out prior to irrigation. Irrigationwas purposely applied directly at the site of the defect, yet the sampledid not migrate after 5 irrigations and was evaluated as a “10 out of10”.

A sample of 16 wt % water soluble chitosan (FMC), 0.3 mm adhesivethickness, Ebeam (DT-86-6) was placed on moist dura, which had beenmoistened with PBS. Sample was held in place for 10 seconds using asingle finger; the sample was wet out prior to irrigation. The samplemigrated after 2 irrigations and was evaluated as a “5 out of 10.”

A sample of 60:40 LMW:HMW chitosan, 0.3 mm adhesive thickness, EU(DT-83-24) was placed on moist dura, which had been moistened with PBS.Sample was held in place for 10 seconds using a single finger; thesample was wet out prior to irrigation. The corner of the sample came upafter 3 irrigations, and the sample did not migrate after 5 irrigationsand was evaluated as a “7 out of 10”.

A sample of 6.5 wt % MMW chitosan, 0.3 mm adhesive thickness, EO(DT-89-20) was placed on moist dura, which had been moistened with PBS.Sample was held in place for 10 seconds using a single finger; thesample was wet out prior to irrigation. The sample migrated after 2irrigations and was evaluated as a “2 out of 10”.

A sample of 6.5 wt % MMW chitosan, 0.3 mm adhesive thickness, EO(DT-89-1) was placed on moist dura, which had been moistened with PBS.Sample was held in place for 10 seconds using a single finger; thesample was wet out prior to irrigation. The corner of the sample liftedafter 1 irrigation, the majority of the sample lifted after 2irrigations, and the sample migrated after 4 irrigations and wasevaluated as a “3 out of 10”.

The following additional experiments were conducted:

An approximately five month old sample of 5.5 wt % chitosan chloride(FMC) (DT-54-49) was placed on moist dura, which had been moistened withPBS. Sample was held in place for 10 seconds using a single finger; thesample was wet out prior to irrigation. The sample was irrigated fivetimes. The sample provided significant resistance to peel when liftedwith forceps and was evaluated as a “10 out of 10.”

An approximately five month old sample of 5.5 wt % chitosan chloride(FMC) (DT-55-19), which had been moistened with PBS. Sample was held inplace for 10 seconds using a single finger; the sample was wet out priorto irrigation. The sample was irrigated five times. The sample providedsignificant resistance to peel when lifted with forceps and wasevaluated as a “10 out of 10.”

An approximately five month old sample of 18 wt % LMW chitosan(DT-71-13) was placed on moist dura, which had been moistened with PBS.Sample was held in place for 10 seconds using a single finger; thesample was wet out prior to irrigation. The edge of the sample liftedafter the second irrigation. The sample provided moderate resistance topeel when lifted with forceps and was evaluated as a “8 out of 10”.

A sample of 18 wt % LMW chitosan (DT-71-31) was placed on moist dura,which had been moistened with PBS. Sample was held in place for 10seconds using a single finger; the sample was wet out prior toirrigation. The sample was irrigated five times. The sample providedsignificant resistance to peel when lifted with forceps and wasevaluated as a “10 out of 10”.

A sample of 18 wt % LMW chitosan (DT-71-32) was placed on moist dura,which had been moistened with PBS. Sample was held in place for 10seconds using a single finger; the sample was wet out prior toirrigation. The sample was irrigated five times. The sample providedsignificant resistance to peel when lifted with forceps and wasevaluated as a “10 out of 10”.

A sample of 18 wt % LMW chitosan (DT-76-74 (P-4)) was placed on moistdura, which had been moistened with PBS. Sample was held in place for 10seconds using a single finger; the sample was wet out prior toirrigation. The sample was irrigated five times. Half of the samplelifted after the fourth irrigation. The sample provided some resistanceto peel when lifted with forceps and was evaluated as a “9 out of 10”.

A sample of 6.5 wt % MMW chitosan (DT-89-6) was placed on moist dura,which had been moistened with PBS. Sample was held in place for 10seconds using a single finger; the sample was wet out prior toirrigation. The sample lifted at the corner after 1 irrigation andmigrated upon second irrigation and was evaluated as a “3 out of 10”.

A sample of 6.5 wt % MMW chitosan (DT-89-8) was placed on moist dura,which had been moistened with PBS. Sample was held in place for 10seconds using a single finger; the sample was wet out prior toirrigation. The sample began to lift on the third irrigation. The samplemigrated nearly completely after fifth irrigation. The sample provided asmall resistance to peel when lifted the corner that remained stuck tothe dura and was evaluated as a “7 out of 10”.

A sample of (70:30) 18 wt % LMW chitosan: 6.5 wt % MMW chitosan(DT-92-5) was placed on moist dura, which had been moistened with PBS.Sample was held in place for 10 seconds using a single finger; thesample was wet out prior to irrigation. The sample nearly completelymigrated after the second irrigation. The sample migrated completelyafter the third irrigation and was evaluated as a “3 out of 10”.

A sample of (30:70) 18 wt % LMW chitosan: 6.5 wt % MMW chitosan(DT-92-26) was placed on moist dura, which had been moistened with PBS.Sample was held in place for 10 seconds using a single finger; thesample was wet out prior to irrigation. The sample migrated after 1irrigation and was evaluated as a “1 out of 10”.

A sample of 6.5 wt % MMW chitosan (DT-93-5) was placed on moist dura,which had been moistened with PBS. Sample was held in place for 10seconds using a single finger; the sample was wet out prior toirrigation. The sample migrated after the first irrigation and wasevaluated as a “1 out of 10”.

A sample of 18 wt % LMW chitosan (DT-94-1) was placed on moist dura,which had been moistened with PBS. Sample was held in place for 10seconds using a single finger; the sample was wet out prior toirrigation. The first irrigation lifted up the edge of the sample. Thesample did not completely migrate after 5 irrigations, and there was aslight pull on the dura when the remaining corner of the sample waspeeled loose from the dura and was evaluated as a “7 out of 10”.

A sample of 18 wt % LMW chitosan (DT-94-2) was placed on moist dura,which had been moistened with PBS. Sample was held in place for 10seconds using a single finger; the sample was wet out prior toirrigation. The sample lifted on the third irrigation and was half offafter 5 irrigations. There was a slight pull on the dura when the samplewas peeled loose from the dura and was evaluated as a “8 out of 10”.

A sample of 18 wt % LMW chitosan (DT-94-3) was placed on moist dura,which had been moistened with PBS. Sample was held in place for 10seconds using a single finger; the sample was wet out prior toirrigation. The sample did not migrate after five irrigations. There wasa significant pull on the dura when the sample was peeled loose from thedura and was evaluated as a “10 out of 10”.

A sample of thicker layered 18 wt % LMW chitosan (DT-94-141) was placedon moist dura, which had been moistened with PBS. Sample was held inplace for 10 seconds using a single finger; the sample was wet out priorto irrigation. Half of the sample lifted after the second irrigation,but did not migrate after 5 irrigations. There was some pull on the durawhen the sample was peeled loose from the dura and was evaluated as a “7out of 10”.

A sample of thicker layered 18 wt % LMW chitosan (DT-94-142) was placedon moist dura, which had been moistened with PBS. Sample was held inplace for 10 seconds using a single finger; the sample was wet out priorto irrigation. The sample was placed close to the dural defect whichyielded the sample getting wet out during the hold. The sample did notmigrate after five irrigations. There was some pull on the dura when thesample was peeled loose and was evaluated as a “7 out of 10”.

A sample of 18 wt % LMW chitosan (DT-94-143) was placed on moist dura,which had been moistened with PBS. Sample was held in place for 10seconds using a single finger; the sample was wet out prior toirrigation. The sample's edge lifted after the fifth irrigation. Therewas some pull on the dura when the sample was peeled loose and wasevaluated as a “9 out of 10”.

A sample of thicker layered 18 wt % LMW chitosan (DT-94-142) was placedon moist dura, which had been moistened with PBS. Sample was held inplace for 10 seconds using a single finger; the sample was wet out priorto irrigation. The sample's edge began to lift after the secondirrigation, but did not migrate after five irrigations. There was somepull on the dura when the sample was peeled loose and was evaluated as a“9 out of 10”.

A sample of (70:30) 18 wt % LMW chitosan: 6.5 wt % MMW chitosan(DT-95-1) was placed on moist dura, which had been moistened with PBS.Sample was held in place for 10 seconds using a single finger; thesample was wet out prior to irrigation. The sample did not migrate afterfive irrigations, but lifted at the edge on the fifth irrigation. Therewas some pull on the dura when the sample was peeled loose and wasevaluated as a “10 out of 10”.

A sample of thicker layered (70:30) 18 wt % LMW chitosan: 6.5 wt % MMWchitosan (DT-95-2) was placed on moist dura, which had been moistenedwith PBS. Sample was held in place for 10 seconds using a single finger;the sample was wet out prior to irrigation. The sample did not migrateafter five irrigations, but lifted at the edge on the fourth irrigation.The sample provided minimal resistance to peel when lifted off of thedura and was evaluated as a “8 out of 10”:

A sample of (70:30) 18 wt % LMW chitosan: 6.5 wt % MMW chitosan(DT-95-3) was placed on moist dura, which had been moistened with PBS.Sample was held in place for 10 seconds using a single finger; thesample was wet out prior to irrigation. The sample did not migrate afterfive irrigations and had a very significant pull on the dura during thepeel up. Irrigations did not appear to lift the sample at all and wasevaluated as a “10 out of 10”.

A sample of thicker layered (70:30) 18 wt % LMW chitosan: 6.5 wt % MMWchitosan (DT-95-61) was placed on moist dura, which had been moistenedwith PBS. Sample was held in place for 10 seconds using a single finger;the sample was wet out prior to irrigation. The sample did not migrateafter 5 irrigations, but began to lift on the third irrigation. A peeltest demonstrated some pull against the dura and was evaluated as a “8out of 10”.

A sample of thicker layered (70:30) 18 wt % LMW chitosan: 6.5 wt % MMWchitosan (DT-95-62) was placed on moist dura, which had been moistenedwith PBS. Sample was held in place for 10 seconds using a single finger;the sample was wet out prior to irrigation. The sample began to migrateafter the second irrigation, but did not completely migrate after fiveirrigations. A peel test demonstrated some pull on the dura and wasevaluated as a “7 out of 10”.

A sample of thicker layered (70:30) 18 wt % LMW chitosan: 6.5 wt % MMWchitosan (DT-95-63) was placed on moist dura, which had been moistenedwith PBS. Sample was held in place for 10 seconds using a single finger;the sample was wet out prior to irrigation. The sample's corner liftedon the first irrigation, but did not completely migrate after fiveirrigations. A peel test demonstrated some pull on the dura and wasevaluated as a “7 out of 10”.

A sample of thicker layered 18 wt % LMW chitosan (DT-96-1) was placed onmoist dura, which had been moistened with PBS. Sample was held in placefor 10 seconds using a single finger; the sample was wet out prior toirrigation. The edge of the sample was lifted during the fourthirrigation, but did not migrate after five irrigations. A peel testdemonstrated a slight pull on the dura and was evaluated as a “8 out of10”.

A sample of (30:70) 18 wt % LMW chitosan: 6.5 wt % MMW chitosan(DT-97-1) was placed on moist dura, which had been moistened with PBS.Sample was held in place for 10 seconds using a single finger; thesample was wet out prior to irrigation. The sample edge lifted after thefirst irrigation but did not migrate after five irrigations. A peel testdemonstrated some pull on the dura and was evaluated as a “7 out of 10”.

A sample of (30:70) 18 wt % LMW chitosan: 6.5 wt % MMW chitosan(DT-97-2) was placed on moist dura, which had been moistened with PBS.Sample was held in place for 10 seconds using a single finger; thesample was wet out prior to irrigation. The sample's top edge liftedafter the second irrigation and migrated completely after fourirrigations and was evaluated as a “6 out of 10”.

A sample 6.5 wt % MMW chitosan (DT-98-1) was placed on moist dura, whichhad been moistened with PBS. Sample was held in place for 10 secondsusing a single finger; the sample was wet out prior to irrigation. Thesample was placed close to the dural defect which yielded the samplegetting wet out during the hold. The sample migrated during the secondirrigation and was evaluated as a “3 out of 10”.

A sample of 6.5 wt % MMW chitosan (DT-98-2) was placed on moist dura,which had been moistened with PBS. Sample was held in place for 10seconds using a single finger; the sample was wet out prior toirrigation. The sample began to migrate at the corner after the secondirrigation and completely migrated on the fifth irrigation and wasevaluated as a “7 out of 10”.

A sample of 6.5 wt % MMW chitosan (DT-98-1) was placed on moist dura,which had been moistened with PBS. The sample began to lift after thefourth irrigation but did not completely migrate after the fiveirrigations. A peel test demonstrated slight pull on the dura and wasevaluated as a “8 out of 10”.

A sample of 6.5 wt % MMW chitosan (DT-98-3) was placed on moist dura,which had been moistened with PBS. Sample was held in place for 10seconds using a single finger; the sample was wet out prior toirrigation. The sample edge lifted after the first irrigation, andcompletely migrated after the third irrigation and was evaluated as a “5out of 10”.

Given the benefit of the above disclosure and description of exemplaryembodiments, it will be apparent to those skilled in the art thatnumerous alternative and different embodiments are possible in keepingwith the general principles of the invention disclosed here. Thoseskilled in this art will recognize that all such various modificationsand alternative embodiments are within the true scope and spirit of theinvention. The appended claims are intended to cover all suchmodifications and alternative embodiments. It should be understood thatthe use of a singular indefinite or definite article (e.g., “a,” “an,”“the,” etc.) in this disclosure and in the following claims follows thetraditional approach in patents of meaning “at least one” unless in aparticular instance it is clear from context that the term is intendedin that particular instance to mean specifically one and only one.Likewise, the term “comprising” is open ended, not excluding additionalitems, features, components, etc.

What is claimed is:
 1. An adherent resorbable porous device forregenerating meningeal tissue in a patient, comprising: a biocompatibleand resorbable collagen matrix sheet-having pores of sizes that permitgrowing meningeal tissue to infiltrate therein, and an adhesive layerhaving a thickness between 0.10 mm to 1 mm of dried hydroxypropyl methylcellulose or hydroxyethyl cellulose adhesive on only one surface of thecollagen matrix sheet, which when contacted with aqueous fluid providesadherence to a surface of the meningeal tissue and allows infiltrationof cells of the meningeal tissue into the collagen matrix sheet, whereinthe adhesive layer provides adherence to the surface of the meningealtissue with an increased uniaxial adhesion from about 0.4N to about 10Nas determined by ASTM F-2258-05; and wherein the adherent resorbableporous device has a porous structure that facilitates ingrowth of cellsinto the collagen matrix.
 2. The adherent resorbable porous device ofclaim 1 further including a bioactive agent.
 3. The adherent resorbableporous device of claim 1 wherein the hydroxypropyl methyl cellulose orhydroxyethyl cellulose has a molecular weight between 5 kDa and 2000kDa.
 4. The adherent resorbable porous device of claim 1 wherein thehydroxypropyl methyl cellulose or hydroxyethyl cellulose has a molecularweight between 10 kDa and 1500 kDa.
 5. The adherent resorbable porousdevice of claim 1 wherein the adhesive layer includes a dye coloringagent.
 6. The adherent resorbable porous device of claim 1 wherein thematrix sheet includes a dye coloring agent.
 7. The adherent resorbableporous device of claim 1 wherein the matrix sheet and the adhesive layerinclude a dye coloring agent.
 8. The adherent resorbable porous deviceof claim 7 wherein the matrix sheet and the adhesive layer are differentcolors.
 9. The adherent resorbable porous device of claim 1 wherein thematrix sheet has a thickness from 2.5 mm to 5 mm.
 10. The adherentresorbable porous device of claim 1 wherein the matrix sheet is acollagen sponge.
 11. The adherent resorbable porous device of claim 1wherein the matrix sheet has a thickness from 1.0 mm to 5 mm.
 12. Theadherent resorbable porous device of claim 1 wherein the matrix sheetincludes pores of from 1 μm to 1000 μm.
 13. The adherent resorbableporous device of claim 1 wherein the matrix sheet includes pores of from10 μm to 500 μm.
 14. The adherent resorbable porous device of claim 1,wherein the adhesive layer has a thickness between about 0.2 mm to about0.5 mm.
 15. The adherent resorbable porous device of claim 1, whereinthe adhesive layer has a thickness between about 0.3 mm to about 0.4 mm.16. The adherent resorbable porous device of claim 1, wherein theadhesive layer is formed on the collagen matrix sheet by applying asolution of the adhesive to one surface of the collagen matrix sheetwhen it is dry and then lyophilized.
 17. The adherent resorbable porousdevice of claim 1, which is prepared by evenly applying a solution ofthe adhesive onto one side of a lyophilized collagen matrix sheet, andthen lyophilizing the collagen matrix sheet having the adhesive appliedthereon.